Positive Selection in the Carbohydrate Recognition Domains of Sea Urchin
Sperm Receptor for Egg Jelly (suREJ) Proteins
Silvia A. Mah,* Willie J. Swanson, and Victor D. Vacquier*
*Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego,
La Jolla; and Department of Genome Sciences, University of Washington, Seattle
A wealth of evidence shows that protein-carbohydrate recognition mediates the steps of gamete interaction during
fertilization. Carbohydrate-recognition domains (CRDs) comprise a large family of ancient protein modules of
approximately 120 amino acids, having the same protein fold, that bind terminal sugar residues on glycoproteins and
polysaccharides. Sea urchin sperm express three suREJ (sea urchin receptor for egg jelly) proteins on their plasma
membranes. suREJ1 has two CRDs, whereas suREJ2 and suREJ3 both have one CRD. suREJ1 binds the fucose sulfate
polymer (FSP) of egg jelly to induce the sperm acrosome reaction. The structure of FSP is species specific. Therefore, the
suREJ1 CRDs could encode molecular recognition between sperm and egg underlying the species-specific induction of
the acrosome reaction. The functions of suREJ2 and suREJ3 have not been explored, but suREJ3 is exclusively localized
on the plasma membrane over the sperm acrosomal vesicle and is physically associated with sea urchin polycystin-2,
a known cation channel. An evolutionary analysis of these four CRDs was performed for six sea urchin species.
Phylogenetic analysis shows that these CRDs were already differentiated in the common ancestor of these six sea
urchins. The CRD phylogeny agrees with previous work on these species based on one nuclear gene and several
mitochondrial genes. Maximum likelihood shows that positive selection acts on these four CRDs. Threading the suREJ
CRDs onto the prototypic CRD crystal structure shows that many of the sites under positive selection are on extended
loops, which are involved in saccharide binding. This is the first demonstration of positive selection in CRDs and is
another example of positive selection acting on the evolution of gamete-recognition proteins.
Introduction
Sea urchin spermatozoa (sperm) are model cells for
studying flagellar motility (Gibbons 1996; Brokaw 2002;
Imai and Shingyoji 2003), the acrosome reaction (Darszon
et al. 2001; Neill and Vacquier 2004), chemotaxis toward
egg-released molecules (Ward et al. 1985; Garbers 1989;
Kaupp et al. 2003), and species-specific binding to eggs
(Glabe and Lennarz 1979; Metz et al. 1994; Vacquier,
Swanson, and Hellberg 1995; Kamei and Glabe 2003).
Although many of the proteins mediating these processes
are known in some detail, relatively little is known about
the evolution of sperm proteins involved in the underlying
signal-transduction events.
The acrosome reaction (AR) is a general feature of
animal sperm that renders the sperm capable of penetrating
egg investments and fusing with the egg plasma membrane.
We are studying the sea urchin sperm plasma membrane
proteins that bind egg jelly (EJ) molecules to induce the AR.
The interaction of EJ with sperm causes ion channels
to activate, resulting in the influx of Ca21 and Na1 and the
efflux of K1 and H1. In addition to these ion fluxes, there
is the depolarization of the sperm plasma membrane, an
increase in intracellular pH of approximately 0.25 units,
and increases in cAMP, cGMP, and protein kinase activity
(Garbers 1989; Darszon et al. 2001; Neill and Vacquier
2004).
Monoclonal antibodies (Mabs) were produced that
react with a 210-kDa glycoprotein present on the flagellar
plasma membrane and also on the plasma membrane
covering the sea urchin sperm acrosomal vesicle (Trimmer
Key words: positive selection, fertilization, acrosome reaction,
sperm lectins, sperm receptors, C-type lectins, maximum likelihood,
sexual antagonism.
E-mail: [email protected].
Mol. Biol. Evol. 22(3):533–541. 2005
doi:10.1093/molbev/msi037
Advance Access publication November 3, 2004
1987). Mabs to this protein induce the AR and compete
with EJ for AR induction (Moy et al. 1996). When this
sperm protein was purified and attached to agarose beads,
the fucose sulfate polymer (FSP) of EJ was the only EJ
component binding the beads (Vacquier and Moy 1997).
The FSP component of EJ is an indispensable inducer of
the AR (Vacquier and Moy 1997; Hirohashi and Vacquier
2002a). This 210-kDa protein was named suREJ1 (sea
urchin receptor for egg jelly–1), and cloning it showed that
its NH2-terminal region possessed two carbohydrate
recognition domains (CRDs) of the intron-containing,
calcium-dependent (C-type lectin) variety (Taylor et al.
1990; Drickamer 1999; Drickamer and Fadden 2002;
Taylor and Drickamer 2003).
When suREJ1 was cloned, two additional homologous
cDNAs were obtained and named suREJ2 and suREJ3.
suREJ2 has one N-terminal CRD and appears to be an
intracellular plasma membrane protein located over the
entire cell but concentrated over the giant sperm mitochondrion (Galindo, Moy, and Vacquier 2003a). Functional
studies have not been done on suREJ2. suREJ3 has one
N-terminal CRD and is localized exclusively on the
sperm plasma membrane covering the acrosomal vesicle,
suggesting it may be involved in AR signal transduction
(Mengerink, Moy, and Vacquier 2002; Neill and Vacquier
2004). suREJ3 is physically associated with the sea urchin
homolog of human polycystin-2 (Neill, Moy, and Vacquier
2004), an important human disease protein known to form
nonselective cation channels in mammalian cells (Cantiello
2004; Delmas et al. 2004).
Collectively, the three suREJ plasma membrane
proteins possess a total of four CRDs, which suggests
that they function by binding specific terminal saccharide
residues (Taylor and Drickamer 2003). CRDs are ancient
protein domains (King, Hittinger, and Carroll 2003)
of approximately 120 amino acids, which form a major
portion of the C-type lectin superfamily (Ebner, Sharon,
Molecular Biology and Evolution vol. 22 no. 3 Ó Society for Molecular Biology and Evolution 2004; all rights reserved.
534 Mah et al.
and Ben-Tal 2003) and are important in cell-cell adhesion,
innate immune recognition, and cell signaling. Crystallographic studies show that all CRDs have the same protein
fold (Taylor and Drickamer 2003).
Many eukaryotic gamete recognition proteins exhibit
rapid evolution, which can be driven by positive Darwinian
selection (Swanson and Vacquier 2002). Positive selection
results from fixation of mutations that increase the fitness of
the organism. To determine whether positive selection has
acted on the evolution of the four CRDs of the three suREJ
proteins, we analyzed the four CRD sequences from six sea
urchin species.
Materials and Methods
RNA Isolation
Fresh testes (0.8 g) from Strongylocentrotus purpuratus, S. franciscanus, and Allocentrotus fragilis (all three are
Northeast Pacific species) and testes fixed in 80% ethanol
and stored at 2208C of S. pallidus, S. droebachiensis (both
circumpolar Arctic species), and Hemicentrotus pulcherrimus (Northwest Pacific), were extracted with 8 ml TRIZOL
reagent (Invitrogen) following the manufacturer’s high-salt
procedure. For polymorphism studies of one species, RNA
was isolated from testes of five male S. purpuratus from the
same population.
cDNA Synthesis
First-strand cDNA synthesis was performed using the
Invitrogen Superscript protocol. Reactions contained 2.5
mg total RNA, 200 lg random primers, 4 ll 10 mM dNTP,
and DEPC-treated water to a total volume of 12 ll. Reactions were heated 3 min at 708C and frozen in dry ice–
ethanol. Tubes were thawed and 4 ll of 53 first-strand
buffer, 2 ll 100 lM DTT and 1 ll RNase OUT added and
the tubes incubated 10 min at 238C, and then 2 min at
428C. One ml of Superscript II RT was added and the
tubes incubated 428C for 1 h, followed by 708C for 15 min.
One microliter of RNase H (2U/ll) was added and the
tubes incubated 378C for 20 min before storage at 2208C.
Amplification and Sequencing of suREJ CRDs
Forty-five exact-match primers to the four S.
purpuratus CRD sequences were used to amplify the
CRDs from the three suREJ proteins of the other five sea
urchin species. PCR reactions contained 50 ng template
cDNA or genomic DNA, 1.5 ll 50 mM MgCl2, 5 ll 10X
Taq buffer, 2 ll 10 mM dNTPs (2.5 mM each nucleotide),
0.25 ll Taq polymerase (Bioline), and water to 50 ll total
volume. Temperature cycling used a hot start of 3 min at
958C, followed by 35 cycles of 948C for 1 min, 428C to
608C for 1 to 4 min, and 728C for 1 to 4 min and a final
extension of 728C for 5 min. PCR products were TA
cloned (Invitrogen) or gel extracted (Qiagen) for sequencing. Big dye sequencing (ABI) reactions were performed,
precipitated with ethanol, and PCR products were dried
and sequenced by the UCSD AIDS Center or Sequegene
Inc. (www.sequegene.com). GenBank accession numbers
for the three suREJ proteins of S. purpuratus are U40832
(suREJ1), AY346376 (suREJ2), and AF422153 (suREJ3).
The 20 CRDs of the three suREJ proteins of the other five
species are GenBank numbers AY620378 to AY620397.
Sequence Analysis
Sequencher and MacVector were used to align DNA
and translated sequences. ClustalW was used for multiple
alignments. The PHYLIP program dnamL was used for phylogenetic trees (Felsenstein 2004). A transition/transversion
ratio (¼ 1.9) and other parameters were estimated from
the data. Blast (Altschul et al. 1997) was used to search
the Prosite, Pfam, InterPro, CCD, and SMART databases.
Nonsynonymous (dN) and synonymous (dS) nucleotide
substitutions were calculated using CODEML from the
PAML software package (Yang 1997). For the analysis of
the variation in the dN/dS ratio between sites, we calculated
the likelihood of a neutral model where no codons could
have a dN/dS ratio greater than 1 (L0) and compared it with
the likelihood of a model in which a subset of sites could
have a dN/dS ratio greater than 1 (L1). The negative of
twice the difference in the log-likelihood obtained from
these two models (22[log(L0) 2 log(L1)]) was compared
with the v2 distribution with degrees of freedom equal to
the difference in number of estimated parameters. The
variation in the dN/dS ratio between sites was modeled
using both a discrete (PAML models M0 and M3) and beta
(PAML models M7 and M8) distributions. The M0 versus
M3 results were consistent with the M7 versus M8 results,
but only the latter are presented because it is a more robust
test of adaptive evolution. We checked for convergence by
performing the analyses from different initial dN/dS ratios
(Bielawski and Yang 2003).
Results
Phylogeny of the 24 suREJ CRDs
Alignment of the 24 CRDs of the four suREJ proteins
of the six sea urchin species shows that 18 out of 120
amino acid positions (15%) are identical (fig. 1). The five
Cys residues are completely conserved, as are many
positions occupied by aromatic residues. Sequencing genomic DNA shows the presence of two introns in all CRDs
at positions 49 and 84. The consensus residues of the 28
positions that define intron-containing, calcium-dependent
CRDs (Taylor and Drickamer 2003) are shown in bold
letters above the alignment. Fifteen of these positions
agree with the consensus in at least 16 of the CRDs,
supporting the classification of these CRDs in this large
protein domain family. The largest single block of conserved residues is between positions 34 and 47.
A neighbor-joining tree of the 24 CRD sequences
(fig. 2) shows that with the exception of the S. franciscanus
suREJ2CRD, 23 of the CRDs cluster together as to the type
of CRD and not as to species. The tree topology shows that
the four suREJ CRDs were already differentiated from each
other in the common ancestor of these six species. This is
also supported by the fact that in the six pairwise
comparisons of the four CRDs of S. purpuratus the percent
identity ranges from 40% to 46%, whereas pairwise
comparisons of the same CRD between any two species
Positive Selection in suREJ Proteins 535
FIG. 1.—Alignment of all 24 CRDs of the three suREJ proteins of the six sea urchin species. The consensus sequence for the intron-positive,
calcium-dependent variety of CRDs is shown in bold above the sequence. Arrow heads mark the conserved positions of the two introns. Dashes are
inserted for optimal alignment. Dark gray boxed residues indicate identity in at least 12 sequences. Spu, S. purpuratus; Afr, A. fragilis; Spa, S. pallidus;
Sdr, S. droebachiensis; Hpu, H. pulcherrimus; Sfr, S. franciscanus; R1C1, suREJ1CRD1; R1C2, suREJ1CRD2; R2C, suREJ2CRD; R3C,
suREJ3CRD. These designations are used for all figures.
range from 69% to 97% in suREJ1CRD1, 65% to 97% in
suREJ1CRD2, 45% to 95% in suREJ2CRD, and 67% to
94% in suREJ3CRD (table 1).
Positive Selection in suREJ CRDs
Two methods were used to test for positive selection
(adaptive evolution). First, the average dN/dS ratios across
the entire CRD sequences in all 15 pairwise comparisons of
the six species were calculated (table 1). Plots of dN versus dS
show that for suREJ1CRD2, all 15 comparisons fall above
the line of neutral expectation (fig. 3). Although none of the
pairwise comparisons are significantly different from 1 after
correction for multiple testing (Bonferroni 1936), these
results do indicate the likely action of positive Darwinian
selection driving the divergence of this domain. For the
majority of the other CRD comparisons, the dN/dS ratios are
high (;0.5) compared with average dN/dS ratios of most
genes (;0.2). Many genes with a ratio of 0.5 or higher are in
fact subjected to positive selection (Swanson et al. 2004).
Because it is known that averaging dN/dS ratios across
all sites and lineages is not a robust test for positive selection,
we tested for variation in the dN/dS ratios between sites.
Maximum likelihood was used to identify those sites in each
CRD that are subjected to positive selection with a posterior
probability greater than 0.95. The analysis (table 2) shows
12 sites in suREJ1CRD1, 11 sites in suREJ1CRD2, seven
sites in suREJ2CRD, and nine sites in suREJ3CRD.
Threading suREJCRDs onto the CRD Crystal Structure
Because of the high conservation of Cys and aromatic
residues, all intron-containing, calcium-dependent CRDs
have the same three-dimensional structure (Taylor and
Drickamer 2003). The four CRD amino acid sequences
were threaded onto a known CRD crystal structure
536 Mah et al.
pectations, the amino acid differences potentially result in
functional differences within this species, as has been
observed for polymorphisms in sea urchin sperm bindin, the
protein that binds the sperm to the egg surface (Palumbi
1999).
Discussion
Phylogeny and Rate of CRD Change in the Six Species
FIG. 2.—Neighbor-joining tree of the 24 suREJ CRDs (500 replicas).
Bootstrap values are shown on nodes. The CRDs were already
differentiated from each other before the speciation of these six sea
urchins.
(Feinberg et al. 2000) and the sites subjected to positive
selection marked on each structure (fig. 4). For three of the
CRDs (suREJ1CRD1, suREJ2CRD, and suREJ3CRD),
similar regions appear to be subjected to positive selection.
These include several of the extended loops implicated in
saccharide binding and a region near the N-terminus
(Taylor and Drickamer 2003). In all four CRDs, the sites
predicted to be subjected to positive selection appear to be
located on the front face of the CRD. These sites are likely
to be involved in the species-specific recognition of EJ
carbohydrate polymers of the different species.
Polymorphism in the Four CRDs from the Three
S. purpuratus suREJ Sequences
The four CRDs were sequenced from five S. purpuratus from the same population. suREJ1CRD1 has three
polymorphic nucleotide sites, one of which is nonsynonymous (amino acid altering). suREJ1CRD2 has two
polymorphic sites, both of which are nonsynonymous.
suREJ2CRD has five polymorphic sites, four of which
change the amino acid. suREJ3CRD has six polymorphic
sites, and five are amino acid altering. Of the 16 polymorphic
sites in the five individuals, 12 are nonsynonymous and
three of these are subjected to positive selection. The percent
nucleotide polymorphism varies from 0.56% in suREJ1CRD2 to 1.7% in suREJ3CRD. Although none of these
polymorphism levels depart from equilibrium-neutral ex-
The complete sequences of the three suREJ proteins
are known only for S. purpuratus. Thus, the analysis of
these CRDs reflects only the CRDs themselves (;120
residues) and not the entire proteins, which vary in S.
purpuratus from 1,450 (suREJ1) to 2,681 (suREJ3)
residues. In pairwise comparisons of amino acid position
(table 1), the four S. purpuratus CRDs are 42% to 46%
identical, showing that they have diverged considerably
since they arose by gene duplication. Figure 2 shows
that these four CRDs were already differentiated in the
common ancestor of these six species. The phylogenetic
topology of the 24 CRDs agrees with trees of these species
based on the sperm protein bindin (Biermann 1998) and
several mitochondrial genes (Biermann, Kessing, and
Palumbi 2003; Lee 2003). The major point of agreement
is that S. purpuratus, S. droebachiensis, S. pallidus, and
A. fragilis always fall close to each other, on a different
branch from S. franciscanus and H. pulcherrimus. Thus, as
previously discussed (Biermann 1998; Biermann, Kessing,
and Palumbi 2003; Lee 2003), the genera Allocentrotus
and Hemicentrotus should be within the genus Strongylocentrotus. The only point of disagreement between
figure 2 and the previously published trees is with
S. franciscanus suREJ2CRD, which is so divergent that
it falls on its own branch basal to its five homologs.
Lee (2003) has estimated divergence times for these
six species based on 12S rDNA. Using the averages of
Lee’s range of divergence times and the percent identities
in table 1, we calculated the average percent amino acid
change per million years in the 10 pairwise comparisons of
the species in common between Lee’s study and ours.
Averaging the 10 comparisons yields 2.8% amino acid
divergence per million years for suREJ1CRD1, 2.6%
for suREJ1CRD2, 4.2% for suREJ2CRD, and 1.9% for
suREJ3CRD. These data suggest that suREJ2CRD is the
fastest changing and suREJ3CRD the slowest changing
CRD.
Function of suREJ Proteins
The functions of suREJ2 and suREJ3 remain unknown. However, the location of suREJ3 exclusively in
the plasma membrane over the acrosomal vesicle and the
fact that it is physically associated with polycystin-2,
suggest that it may form a cation channel that functions in
the induction of the AR (Neill, Moy, and Vacquie 2004).
The low cation selectivity of polycystin-2 channels would
explain older data on cation transport into sea urchin sperm
suggesting that calcium and sodium entered through the
same ion channel during the acrosome reaction (Schackmann and Shapiro 1981).
Positive Selection in suREJ Proteins 537
Table 1
Percent Identities and dN/dS in Pairwise Comparisons of CRDs
S. purpuratus CRDs
REJ1CRD1
–
REJ1CRD1
REJ1CRD2
REJ2CRD
REJ3CRD
REJ1CRD2
45
–
REJ2CRD
46
42
–
REJ3CRD
45
40
45
–
%ID REJ1CRD1 above and REJ1CRD2 below the diagonal
Spr
Spr
Afr
Spa
Sdr
Hpu
Sfr
85
78
81
73
70
–
(1.5)
(4.5)
(3.5)
(1.1)
(1.4)
Afr
Spa
Sdr
74 (0.6)
–
88 (3.0)
91 (1.9)
71 (1.3)
69 (1.4)
84 (0.4)
83 (0.7)
–
96 (inf)
67 (1.8)
65 (2.1)
76 (0.5)
85 (4.8)
87 (0.4)
–
70 (1.5)
68 (1.8)
Hpu
72
70
73
70
(0.6)
(0.6)
(0.9)
(0.6)
–
97 (inf)
Sfr
72
69
72
70
97
(0.5)
(0.6)
(0.8)
(0.6)
(0.5)
–
%ID REJ2CRD above and REJ3CRD below the diagonal
Spr
Spr
Afr
Spa
Sdr
Hpu
Sfr
94
91
92
81
75
–
(0.2)
(0.5)
(0.2)
(0.6)
(1.5)
Afr
Spa
Sdr
95 (0.3)
–
87 (0.6)
88 (0.6)
77 (0.4)
72 (0.9)
91 (1.2)
90 (0.4)
–
92 (0.2)
82 (0.4)
74 (1.5)
76 (0.5)
72 (1.2)
74 (3.1)
–
86 (0.6)
71 (1.1)
Hpu
61
58
62
61
(0.5)
(0.6)
(0.7)
(0.2)
–
67 (1.0)
Sfr
47
47
48
50
45
(0.7)
(0.6)
(0.7)
(0.6)
(0.8)
–
NOTE.—The dN/dS ratio is in parentheses. Inf indicates infinity because dS was 0.
Although it has not been experimentally demonstrated, the existence of at least one CRD in the three
suREJ proteins suggests that their function involves binding saccharide residues (Taylor and Drickamer 2003).
Agarose beads with covalently immobilized suREJ1 bind
the FSP component of EJ. FSP is a pure polysaccharide
with no amino acid content and a molecular mass greater
than 1 million Da (Vacquier and Moy 1997; Hirohashi and
Vacquier 2002a). FSP is indispensable for the induction of
the sperm AR, and it can be very species specific as an AR
inducer. This specificity of molecular recognition resides
in the pattern of sulfation of the fucosyl residues and the
nature of the glycosidic bond (Vilela-Silva et al. 2002).
With the exception of suREJ1CRD2, many of the sites
under positive selection in the other three CRDs are
located on the external faces of the extended loops that are
known to bind saccharide residues (Taylor and Drickamer
2003). The evolution of CRDs on sperm receptor proteins
could be one way in which the species specificity of
gamete recognition is encoded.
Monoclonal antibody to suREJ1 induces the AR and
competes with EJ for AR induction (Moy et al. 1996),
showing that suREJ1 plays a major role in AR induction.
Both CRDs of suREJ1 show positive selection, but
FIG. 3.—Plots of dN (vertical axis) versus dS (horizontal axis) for the 15 possible pairwise comparisons of the four CRDs. The method is an average
across the CRD. In suREJ1CRD2, all comparisons fall above the line of neutral expectation.
538 Mah et al.
Table 2
Positive Darwinian Selection in CRDs of Sperm REJ Proteins
Gene
n
Lc
dN/dS
S
22l
REJ1CRD1
6
119
0.67
1.1
11.2
REJ1CRD2
6
120
1.81
0.7
10.2
REJ2CRD
6
117
0.77
2.2
24.5
REJ3CRD
6
117
0.52
0.9
8.0
M8 Parameters
p1
p0
p1
p0
p1
p0
p1
p0
¼
¼
¼
¼
¼
¼
¼
¼
0.27,
0.73,
0.18,
0.82,
0.18,
0.82,
0.26,
0.74,
Positively Selected Sites
dN/dS ¼ 3.1
b(26.8,99.0)
dN/dS ¼ 6.7
b(25.3,.005)
dN/dS ¼ 5.6
b(0.48,0.31)
dN/dS ¼ 2.0
b(18.4,99.0)
F2, L12, V16, D17, Y55, Y72, K86, K90, S92, L103, K116, A117
G17, G18, S21, L36, M51, K105, T106, D116, D117, V119, I122
A13, R16, P19, Q86, N93, K113, H117
E16, N43, S53, S89, H90, F99, N101, S11, N117
NOTE.—The data have n sequences, each of Lc codons. dN/dS is the average ratio over all sites and branches, and S is the tree length in substitutions per codon. M8
parameters include proportion of sites predicted to be under positive selection with their average dN/dS ratio, and the proportion of sites with a dN/dS in the beta interval (b)
between 0 and 1. Positively selected sites with posterior probabilities greater than 0.95 are listed with numbering according to the alignment in figure 1 (with amino acid
indicated according to the S. purpuratus species from each CRD).
selection is more intense on suREJ1CRD2 (fig. 3). This
CRD could play a more significant role in recognizing FSP
as the sperm swims into the hydrated EJ layer. Although FSP
is an indispensable inducer of the AR, another saccharide,
a polysialic acid, is a strong potentiator of the FSP-induced
AR. By itself, the polysialic acid has no AR-inducing
activity; however, in the presence of FSP, it causes an
elevation of intracellular pH. This additional biologically
active saccharide component of EJ must bind to a CRD
carried by a sperm receptor protein (Hirohashi and Vacquier
2002b).
Adaptive Evolution at Several Levels of the Fertilization
Cascade
Sperm-egg interaction proceeds through a series of
steps that are common to most animals (Vacquier 1998).
Sperm may be chemotactically attracted to the egg.
Depending on the species, sperm binds to the egg envelope
either before or after induction of the AR. After exocytosis
of the acrosomal contents, a hole is created in the egg
envelope through which the sperm passes to fuse with the
egg cell membrane. In different animal species, the genes
mediating these steps have been shown to be subjected to
positive selection. Before this study, acrosomal bindin was
the only sea urchin fertilization protein known to be
subjected to positive selection (Metz and Palumbi 1996).
Here, we demonstrate positive selection acting on at least
one gene (suREJ1) that is known to bind an egg carbohydrate (FSP), mediating AR induction. It will be important
to study the pattern of evolution of all genes mediating the
steps in the fertilization cascade to gain insight into the
number of loci that may be involved in establishing
prezygotic reproductive isolation (Coyne and Orr 2004).
Sexual Antagonism As a Reason for Positive Selection
on suREJ CRDs
FIG. 4.—Threading the suREJ CRDs onto the prototypic CRD
crystal structure. Amino acid sites predicted to be under positive selection
are shown in spacefill. Many of the positively selected sites are in similar
regions in the different CRDs.
Our favored hypothesis to explain positive selection in
the evolution of suREJCRDs combines sexual antagonism
(Rice 1996; Gavrilets 2000; Civetta 2003) and sperm
competition (Parker 1970; Birkhead 1996; Frank 2000). In
sexual antagonism (as applied to this paper), sperm and egg
have different ‘‘interests’’ in the efficiency of their
interaction leading to fusion. In presenting this hypothesis,
we must discuss the problem of more than one sperm fusing
with the egg, the biochemistry of the egg jelly coat, the
physical properties of the egg jelly coat, sperm competition,
and positive selection on sperm suREJ proteins.
In sea urchins and mammals, if more than one sperm
fuses with the egg, the result is pathological polyspermy,
Positive Selection in suREJ Proteins 539
which arrests development. Eggs must evolve ways to
decrease the probability of fusion with more than one
sperm. In sea urchins, there is both a fast electrical block to
prevent sperm fusion and a slow physical block that
involves the elevation of a fertilization envelope to prevent
sperm passage. As discussed below, the egg may also
regulate sperm entry through molecular interactions
involving gamete recognition proteins.
When sea urchin eggs are spawned into seawater, the
egg jelly coat (EJC) swells to approximately one egg
diameter and is dense enough to exclude colloidal particles
of India ink. The EJC contains two types of carbohydrate
molecules that are known to be involved in AR induction;
one is FSP and the other is the polysialic acid. In
S. purpuratus (Vacquier and Moy 1997; Alves et al.
1998) and S. droebachiensis (Vilela-Silva et al. 2002), FSP
comes in two isoforms, each isoform being female specific
and both isoforms having equal potency as AR inducers.
(Roughly 64% of S. purpuratus females have one FSP
isoform, 31% have another FSP isoform, and 5% have both
FSP isoforms [Vacquier, unpublished data]). Of the four
other species studied in this report, the structure of FSP is
known for S. franciscanus and S. pallidus. Females of these
two species synthesize only one, species-specific, type of
FSP (Vilela-Silva et al. 2002). The polysialic acid molecule
of the EJC must bind to a sperm-surface receptor. In the
presence of FSP, it greatly potentiates the FSP-induced AR
by raising sperm intracellular pH (Hirohashi and Vacquier
2002b). Thus, in S. purpuratus and S. droebachiensis, there
are at least three types of carbohydrate polymers in the EJC
that interact with sperm-membrane carbohydrate-recognition proteins (suREJ proteins) to regulate sperm ion
channels. The EJC also contains at least 10 other
glycoproteins of unknown function that could bind suREJ
and other unknown sperm-surface receptors (Vacquier and
Moy 1997).
The physical function of the hydrated EJC is to
prevent eggs from sticking together and also to prevent
microbes from binding to the egg surface. If the EJC is
removed from S. franciscanus eggs, the eggs irreversibly
clump together and abnormal development occurs (Vacquier, unpublished data). However, this is not true of S.
purpuratus eggs. In some species of free-spawning echinoderms, the hydrated EJC increases the effective diameter
of the egg, creating a larger target for the sperm to hit.
Thus, in some species, the EJC increases the rate of
fertilization by being of greater diameter than the egg’s
cytoplasmic mass (Levitan and Irvine 2001; Podolsky
2002). In S. purpuratus, eggs in which the bulk of the EJC
has been removed bind more sperm and are more susceptible to higher levels of polyspermy than eggs with
intact EJC. In these ‘‘dejellied’’ eggs, the optimal amount
of EJ needed to induce the sperm AR remains tightly
bound to the egg surface (Vacquier, Brandriff, and Glabe
1979).
From the egg’s side of the sexual antagonism theory,
the hydrated EJC provides three positive things for the
egg: it protects the egg from physical abuse and microbes,
it holds an excess of molecules needed to induce the sperm
AR, and it slows sperm as they approach the egg surface,
by inducing premature AR, which decreases the frequency
of polyspermy (Vacquier, Brandriff, and Glabe 1979;
Frank 2000). Premature induction of the AR decreases
sperm fertilizability with a half-life of 23 seconds
(Vacquier 1979). The EJC slowing the frequency of sperm
fusion is of positive benefit to the egg, but is a negative
benefit for sperm, supporting the hypothesis of sexual
antagonism acting during sperm-egg interaction.
Competition among individual sperm cells will
continuously select for sperm that most efficiently fuse
with eggs (Frank 2000). One ml of undiluted S. purpuratus
semen contains 40 billion sperm cells, and one large male
can spawn 5 ml of semen (Vacquier 1986). Thus, the large
numbers of sperm available enhance the probability that
favorable mutants (in suREJ proteins) will enjoy increased
fertilization success. The egg must evolve ways to retard
these potentially supersuccessful sperm and optimize the
chance for monospermy.
Finally, the suREJ protein variants that optimize sperm
fusion rates with eggs could be selected for on the basis of
changes in their CRDs. Theory suggests that positive
selection should be found in both the sperm protein and its
cognate egg-surface receptor (Gavrilets 2000). Such is the
case with abalone sperm lysin and its egg receptor VERL,
where both cognate binding partners are subjected to
positive selection (Galindo, Vacquier, and Swanson
2003b). The only molecules we know of in sea urchin EJ
that induce the AR in seawater at pH 8.0 are the carbohydrate
polymers of FSP and polysialic acid. Although we do not yet
know how to detect positive selection in a carbohydrate
polymer, it could be that the enzymes involved in the
biosynthesis of these EJ molecules might show positive
selection.
Avoiding polyspermy is a multistep process for the sea
urchin egg. The first level of blockade could be the
interaction of sperm suREJ proteins with egg FSP and
polysialic acid to induce premature AR and retard the rate of
sperm reaching the egg surface. The egg would be selecting
for a sperm that undergoes the AR at exactly the correct
distance from the egg surface (cryptic female choice). The
second level of polyspermy blockade is the well-known
electrical block, where, after fusion with the first sperm, the
egg membrane potential changes in 100 milliseconds from
270 mV to 120 mV, which prevents further sperm fusion
by an unknown mechanism (Gould-Somero and Jaffe
1984). The third polyspermy blockade is the complete
block provided by the fertilization protease and the
formation of the fertilization envelope (Vacquier, Tegner,
and Epel 1973; Haley and Wessel 2004).
The demonstration of positive selection in the
CRDs of suREJ proteins shows once again that the evolution of the molecules and mechanisms involved in
sperm-egg interaction is extremely complex yet always
fascinating.
Acknowledgments
We thank Gary W. Moy and Anna T. Neill for helpful
advice. This work was supported by an NSF Minority
Graduate Fellowship to S.A.M., NIH Grants HD12986 to
V.D.V., and HD42563 to W.J.S.
540 Mah et al.
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Accepted October 26, 2004